Iplex platform

We selected independent SNPs with PCA-adjusted and/or unadjusted P values less than 1.0 × 10⁻⁵ for the replication study. Genotyping for replication was performed using the iPLEX platform (Sequenom, San Diego, CA, Supplementary Data 6). To exclude possible genotyping error, the genotype cluster plots of the selected SNPs for the discovery and replication stages were generated and visually inspected (Supplementary Figures 9 and 10).

Polymorphisms of complement pathway genes were investigated using the Sequenom iPLEX platform. PCR and primer extension oligonucleotides were designed using the My Sequenom Online Tools (Sequenom Inc., San Diego, CA, USA), with a target PCR fragment length of between 80 and 120 bp and target primer extension oligonucleotide of between 15 and 30 bases, according to the manufacturer’s protocol. Oligonucleotide sequences are shown in S2 Table. Shrimp alkaline phosphatase was used to neutralise unincorporated dNTPs after the PCR reaction and the iPLEX Gold resin used for final conditioning after primer extension. The reaction product was nanodispensed onto an array chip by technical staff in the Queen’s University Belfast Genome Core using the MassARRAY nanodispenser prior to operation of the MassARRAY mass spectrometer. Genotyping was then carried out using TYPER software (Sequenom). Each SNP cluster plot was inspected individually for quality of clustering and the mass spectrometry plot examined for potentially erroneous base calls, which were recalled or set to missing if a problem was observed. Data were exported from TYPER and converted to PLINK format using Microsoft Excel 2010.

The genotyping for replication I was performed using the iPLEX platform (Sequenom, San Diego, CA), and replications II and III were performed using the ligation detection reaction method30 (link)31 (link), with technical support from the Shanghai Biowing Applied Biotechnology Company.

Genomic DNA (gDNA) was extracted from blood samples using Nucleon BACC2 DNA extraction kits [Gen-Probe Life Sciences, Tepnel Research Products and Services, Manchester, UK]. Five nano-grammes of gDNA was whole-genome amplified by primer-extension pre-amplification (PEP) using N15 primers (Sigma, UK) and Biotaq (Bioline, UK) polymerase as previously described by Zhang et al. [17 (link)]. Sixty-eight single nucleotide polymorphisms (SNPs) located in the G6PD gene and its flanking regions were selected from the many hundreds of SNPs identified in the literature and on public databases based on the given criteria (1) previous associations with malaria, (2) predicted functional consequences with respect to G6PD enzyme activity (3) estimated minor allele frequency and (4) whether they made a viable Sequenom assay design [18 (link)]. Three gender-typing markers were also included. Assays were performed using the Sequenom® iPLEX platform according to manufacturer’s instruction using diluted PEP DNA (1:10). Genotype calls were made using the Sequenom® Typer v4.03 software [19 (link)].

Human patient-derived melanoma cell lines were cultured in DMEM medium supplemented with 5% fetal bovine serum and grown at 37°C in 5% CO2. All cell lines were periodically authenticated by DNA finger printing using Life Technologies AmpFISTR Identifier microsatellite kit and tested for mycoplasma by Lonza Mycoalert Assay. Genomic DNA was analyzed for mutations in BRAF, PTEN, NRAS, KIT, CDKN2A, RB, TP53 by the nucleotide extension assay using the iPlex platform (Sequenom, Inc, San Diego, CA) as previously described [40 (link)]. RO4929097, PLX4032, PD0325901, AZD8055, GDC0941, BEZ235 were obtained from SelleckChem (Houston, TX). Cell Viability was determined using MTS assays as previously described [32 ]. IC50 values and maximum inhibitory activity (Amax) were calculated using GraphPad Prism 5 (GraphPad Software Inc.). Amax is defined as the maximal activity of the compound and measures its efficacy in reducing viability.